[D-Phe6 ]-LH-RH and intermediates therefor

ABSTRACT

The decapeptide [D-Phe 6  ]-LH-RH, salts thereof, and intermediates used for the synthesis thereof are disclosed. The decapeptide has potent LH- and FSH-releasing hormone properties.

The invention described herein was made in the course of work under agrant or award from the Department of Health, Education and Welfare.

BACKGROUND OF THE DISCLOSURE

a. Field of the Invention

This invention relates to the decapeptide(pyro)Glu-His-Trp-Ser-Tyr-D-Phe-Leu-Arg-Pro-Gly-NH₂, salts thereof andintermediates for the synthesis thereof.

The decapeptide of this invention also is calledL-pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-phenylalanyl-L-leucyl-L-arginyl-L-prolylglycinamideand may be designated by the abbreviation [D-Phe⁶ ]-LH-RH.

b. Background of the Invention

Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are bothgonadotrophic hormones elaborated by the pituitary gland of humans andof animals. LH together with FSH stimulates the release of estrogensfrom the maturing follicles in the ovary and induces the process ofovulation in the female. In the male, LH stimulates the interstitialcells and is for that reason also called interstitial cell stimulatinghormone (ICSH). FSH induces maturation of the follicles in the ovary andtogether with LH, plays an important role in the cyclic phenomena in thefemale. FSH promotes the development of germinal cells in the testes ofthe male. Both LH and FSH are released from the pituitary gland by theaction of LH- and FSH-releasing hormone, and there is good evidence thatsaid releasing hormone is elaborated in the hypothalamus and reaches thepituitary gland by a neurohumoral pathway, see e.g., A. V. Schally, etal., Recent Progress in Hormone Research, 24, 497 (1968).

The natural LH- and FSH-releasing hormone has been isolated from pighypothalami and its constitution elucidated by A. V. Schally, et al.,Biochem. Biophys. Res. Commun., 43, 393 and 1334 (1971), who proposedthe decapeptide structure(pyro)-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂.

This constitution has been confirmed by snythesis; for example, see H.Matsuo, et al., Biochem. Biophys. Res. Comm., 45, 822 (1971) and R.Geiger et al., ibid, 45, 767 (1971).

Hereinafter the natural LH- and FSH-releasing hormone is called LH-RH.

Because of the importance of LH-RH to both diagnostic and therapeuticmedicine, considerable interest has been shown in the preparation of newcompounds having improved properties over the natural hormone. Oneapproach to this goal has been the replacement of an amino acid residueof LH-RH with another amino acid. Although in a few instancesdecapeptides containing such a replacement have been found to be moreactive than LH-RH, for example, [D-Ala⁶ ]-LH-RH, A. Arimura, et al.,Endocrinology, 95, 1174 (1974) and [D-Leu⁶ ]-LH-RH, J. A.Vilchez-Martinez, et al., Biochem. Biophys. Res. Commun., 59, 1226(1974), for the most part the replacement containing decapeptides havebeen less active.

Now it has been found that the replacement of the glycyl moiety inposition 6 of LH-RH with D-phenylalanine gives a compound that is muchmore active and longer acting than LH-RH. Such attributes of the presentdecapeptide have practical significance: the lesser minimum effectivedose reducing side effects as well as the cost for the preparation ofthe compound and the longer acting property reducing the need forfrequent administration.

SUMMARY OF THE INVENTION

The compounds of this invention are selected from the group consistingof (pyro)-Glu-His-Trp-Ser-Tyr-D-Phe-Leu-Arg-Pro-Gly-NH₂ (l) or anon-toxic, pharmaceutically acceptable salt thereof, and R⁶-(pyro)-Glu-His(N^(lm) -R⁵)-Trp-Ser(R⁴)-Tyr(R³)-D-Phe-Leu-Arg(N^(G)-R²)-Pro-Gly-R^(l) (2), in which R^(l) is selected from the groupconsisting of amino and O- (lower alkyl), R², R³, R⁴ and R⁵ areprotective groups capable of being removed by one or more chemicaltreatments which do not affect(pyro)-Glu-His-Trp-Ser-Tyr-D-Phe-Leu-Arg-Pro-Gly-NH₂ and R⁶ is hydrogenor said protective group.

With reference to R⁶ -(pyro)-Glu-His(N^(lm)-R⁵)-Trp-Ser(R⁴)-Tyr(R³)-D-Phe-Leu-Arg(N^(G) -R²)-Pro-Gly-R^(l), in apreferred embodiment R^(l) is as defined herein, R² is a protectivegroup for the N.sup.δ, N.sup.ω, and N.sup.ω^(') nitrogen atoms ofarginine selected from the group consisting of tosyl, nitro,benzyloxycarbonyl and adamantyloxycarbonyl, R³ is a protective group forthe hydroxyl of tyrosine selected from the group consisting of2-bromo-benzyloxycarbonyl, benzyl, acetyl, tosyl, benzoyl, t-butyl,tetrahydropyran-2-yl, trityl, 2,4-dichlorobenzyl and benzyloxycarbonyl;R⁴ is a protective group for the hydroxyl group of serine and isselected from the group defined hereinbefore for R³ ; R⁵ is a protectivegroup for the imidazole nitrogen atoms of histidine selected from thegroup of tosyl, dinitrophenyl,2,2,2-trifluoro-l-benzoyloxycarbonylaminoethyl and2,2,2-trifluoro-l-t-butyloxycarbonylaminoethyl; and R⁶ is hydrogen or anα-amino protective group selected from the group consisting oft-butyloxycarbonyl, benzyloxycarbonyl, cyclopentyloxycarbonyl,t-amyloxycarbonyl and d-isobornyloxycarbonyl.

A further aspect of the present invention relates to intermediateslinked to a solid resin support. These intermediates are represented bythe formulae:

R⁶ -(pyro)-Glu-His-(N^(lm) -R⁵)-Trp-Ser(R⁴)-Tyr(R³)-D-Phe-Leu-Arg(N^(G)-R²)-Pro-Gly-A,

R⁷ -his-(N^(lm) -R⁵)-Trp-Ser(R⁴)-Tyr(R³)-D-Phe-Leu-Arg(N^(G)-R²)-Pro-Gly-A,

R⁷ -trp-Ser(R⁴)-Tyr(R³)-D-Phe-Leu-Arg(N^(G) -R²)-Pro-Gly-A,

R⁷ -ser(R⁴)-Tyr(R³)-D-Phe-Leu-Arg(N^(G) -R²)-Pro-Gly-A,

R⁷ -tyr(R³)-D-Phe-Leu-Arg(N^(G) -R²)-Pro-Gly-A and

R⁷ -d-phe-Leu-Arg(N^(G) -R²)-Pro-Gly-A

in which R², R³, R⁴, R⁵ and R⁶ are as defined herein and R⁷ is anα-amino protective group known to be useful in the art for the stepwisesynthesis of polypeptides, suitable groups being listed hereinafter, andA is an anchoring bond used in solid phase synthesis linked to a solidresin support. A is selected from the class consisting of: ##STR1##

DETAILS OF THE INVENTION

The term "lower alkyl" contemplates alkyl radicals containing from oneto three carbon atoms and includes methyl, ethyl, propyl and isopropyl.

N^(g) means the side chain nitrogen atoms of arginine.

N^(lm) means the imidazole nitrogen atoms of histidine.

The symbol φ means "phenyl".

In general the abbreviations used herein for designating the amino acidsand the protective groups are based on recommendations of the IUPAC-IUBCommission on Biochemical Nomenclature, see Biochemistry 11, 1726(1972). For instance, t-Boc represents t-butyloxycarbonyl, Z representsbenzyloxycarbonyl, Tos represents tosyl, 2-Br-Cb2 represents2-bromo-benzyloxycarbonyl and Bzl represents benzyl. The abbreviationsused herein for the various amino acids are Arg, arginine; Gly, glycine;His, histidine; Leu, Leucine; Phe, Phenylalanine; Pro, proline;(pyro)-Glu, 5-oxoproline(pyroglutamic acid); Ser, serine; Trp,tryptophan; and Tyr, tyrosine. All amino acids described herein are inthe L-series unless stated otherwise, i.e., D-Phe is a D-phenylalanylresidue.

The polypeptide of this invention can be obtained in the form of an acidaddition salt. Examples of salts are those with organic acids, e.g.acetic, lactic, succinic, benzoic, salicylic, methanesulfonic ortoluenesulfonic acid, as well as polymeric acids such as tannic acid orcarboxymethyl cellulose, and salts with organic acids such as hydrohalicacids, e.g. hydrochloric acid, or sulfuric acid, or phosphoric acid. Ifdesired a particular acid addition salt is converted into another acidaddition salt, e.g. a salt with a non-toxic, pharmaceutically acceptableacid, by treatment with the appropriate ion exchange resin in the mannerdescribed by R. A. Boissonnas, et al., Helv. Chim. Acta, 43, 1349(1960). Suitable ion exchange resins are cellulose based cationexchangers, for example carboxymethylcellulose or chemically modified,cross linked dextran cation exchangers, for example, those of theSephadex C-type, and strongly basic anion exchange resins, for examplethose listed by J. P. Greenstein and M. Winitz in "Chemistry of theAmino Acids", John Wiley and Sons, Inc., New York and London, 1961, Vol.2, p. 1456.

The decapeptide of this invention and its salts possess valuable,long-acting LH- and FSH-releasing hormone activity.

the valuable LH- and FSH-releasing hormone activity and long actingproperty of the compound of this invention are demonstrated by standardpharmacological procedures. For example, these activities can bedemonstrated by tests described by A. Arimura, et al., Endocrinology,95, 1174 (1974). More specifically, by following the procedure describedtherein, LH-release data obtained from rats given equal doses (50 ng,subcutaneously) show that [D-Phe⁶ ]-LH-RH reaches peak activity at abouttwo hours after dosing and that significant activity is still present upto 6 hours; whereas After an injection of LH-RH, peak activity isreached at about the 15 minute mark and no effects of the injection areobserved after one hour. Also integrated levels of LH over a six hourperiod indicate that [D-Phe⁶ ]-LH-RH is about 10 times more active inreleasing LH than LH-RH. FSH-release data following injection of the twocompounds indicate that [D-Phe⁶ ]-LH-RH is about 20 times more activethan LH-RH at the same dose (50 ng). It will be readily appreciated thata compound that is able to effectively release FSH has many therapeuticapplication; see, for example, H. G. Dahlen, et al., Horm. Metab. Res.,6, 510 (1974).

The LH- and FSH-releasing properties of the compound of this invention,which in turn induce ovulation in animals, make the hormone useful inveterinary practice and in animal husbandry. It is often desirable tosynchronize estrus in livestock, for example, cattle, sheep or swine,either in order to be able to mate all the females in a given group witha male of the desired genetic quality, or so as to be able to performartificial insemination on a maximum number of females, both within acomparatively short period of time. In the past, this has been done byadministering to the animals an ovulation-inhibiting agent, withdrawingadministration of said agent shortly before the date chosen for matingor artificial insemination, and relying either upon the naturalproduction of LH and FSH to induce ovulation and to produce estrus or byadministering gonadotrophins. However, this procedure was not entirelysatisfactory because ovulation at a predetermined time occurred never inall the animals together but only in a certain proportion thereof whengonadotrophins were not used. On the other hand, the high cost ofgonadotrophins and side effects encountered in their administration madethis method impractical. It is now possible to obtain substantiallycomplete synchronization of ovulation and of estrus, by treating theanimals in a given group first with an ovulation inhibitor which issubsequently withdrawn, and then administering [D-Phe⁶ ]-LH-RH shortlybefore the predetermined period of time for mating or artificialinsemination, so as to obtain ovulation and estrus within that timeinterval. The delay in the onset of ovulation and estrus followingadministration of [D-Phe⁶ ]-LH-RH varies with the species of animals,and the optimal time interval has to be chosen for each species. Forexample, in rodents such as rats or hamsters ovulation takes placewithin 18 hours following administration of the decapeptide of thisinvention.

The method described above for obtaining ovulation and estrus within aprecisely predetermined time interval, so as to be certain of asuccessful mating, is particularly important for breeders of race horsesand of show animals, where the fees paid for the services of anexceptional male animals often amount to very considerable sums ofmoney.

[D-Pha⁶ ]-LH-RH is also useful to increase the number of live births perpregnancy in livestock, for example, cattle, sheep or swine. For thispurpose the decapeptide is given in a series of parenteral doses,preferably by intravenous or subcutaneous injections, in the range of0.1 - 10 mcg. per kilogram of body weight per day, 96 to 12 hours priorto expected estrus and subsequent mating. A priming injection of 1000 to5000 iu of pregnant mares serum gonadotrophin may also be given one tofour days prior to the above injection of the decapeptide. A similartreatment, with or without prior priming, is also useful for inducingpuberty in farm animals.

When the decapeptide is employed for the purpose of inducing ovulationand esrus or for inducing puberty in warmblooded animals, especially inrodents such as rats or hamsters or in livestock, it is administeredsystemically, preferably parenterally, in combination with apharmaceutically acceptable liquid or solid carrier. The proportion ofthe decapeptide is determined by its solubility in the given carrier, bythe chosen route of administration, and by standard biological practice.For parenteral administration to animals the decapeptide is used in asterile aqueous solution which may also contain other solutes such asbuffers or preservatives, as well as sufficient pharmaceuticallyacceptable salts or glucose to make the solution isotonic. The dosagewill vary with the form of administration and with the particularspecies of animal to be treated and is preferably kept at a level offrom 0.1 mcg. to 10 mcg. per kilogram body weight. However, a dosagelevel in the range of from about 1 mcg. to about 5 mcg. per kilogrambody weight is most desirably employed in order to achieve effectiveresults.

The decapeptide may also be administered in one of the long-acting,slow-release or depot dosage forms described below, preferably byintramuscular injection or by implantation. Such dosage forms aredesigned to release from about 0.1 mcg. to about 10 mcg. per kilogrambody weight per day.

[D-Phe⁶ ]-LH-RH is also useful in human medicine. For example, humanchorionic gonadotrophin (HCG) which contains mainly LH and some FSH hasbeen used for over 30 years to treat certain endocrinological disorderssuch as disturbances of the cycle, amenorrhea, lack of development ofsecondary sex characteristics, and infertility in the female, or certaincases of hypogonadism delayed puberty, crytorchidism, andnon-psychogenic impotence in the male. Lately, infertility in the humanfemale has also been treated with human menopausal gonadotrophin (HMG)which contains mainly FSH, followed by treatment with HCG. One of thedisadvantages of the treatment of infertility in the human female withHCG or with HMG followed by HCG has become apparent in that suchtreatment often results in superovulation and unwanted multiple births,probably because of the impossibility of giving only the exact amountsof FSH and LH which are necessary for ovulation. The administration ofthe decapeptide of this invention overcomes the above disadvantage,because the compound causes release of LH and FSH by the pituitary onlyin the exact quantities which are required for normal ovulation. Forthat reason the decapeptide of this invention is not only useful for theabove purpose, but it is equally useful in the human female in thetreatment of disturbances of the cycle, of amenorrhea, of hypogonadism,and of lack of development of secondary sex characteristics.

Furthermore, the decapeptide of this invention is useful incontraception. For example, when the decapeptide is administered to ahuman female early in the menstrual cycle LH is released at that timeand causes premature ovulation. The immature ovum is either not capableof being fertilized, or, if fertilization should nevertheless have takenplace, it is highly unlikely that the fertilized ovum will becomeimplanted because the estrogen-progestin balance required to prepare theendometrium is not present and the endometrium is not in the conditionnecessary for implantation. On the other hand, when the decapeptide isadministered towards the end of the cycle the endometrium is disruptedand menstruation takes place.

In addition, the decapeptide of this invention is also useful incontraception by the "rhythm" method, which has always been relativelyunreliable because of the impossibility of predetermining ovulation inthe human female with the required degree of accuracy. Administration ofthe decapeptide at mid-cycle, i.e. at about the normally expected timefor ovulation, induces ovulation shortly thereafter and makes the"rhythm" method both safe and effective.

The decapeptide is also useful as a diagnostic tool for distinguishingbetween hypothalamic and pituitary malfunctions or lesions in the humanfemale. When administering the decapeptide to a patient suspected ofsuch malfunctions or lesions and a rise in the level of LH issubsequently observed there is good indication to conclude that thehypothalamus is the cause of the malfunction and that the pituitary isintact. On the other hand, when no rise in circulating LH is seenfollowing the administration of the decapeptide a diagnosis of pituitarymalfunction or lesion can be made with a high degree of confidence.

In the human male, administration of decapeptide provides the amounts ofLH (or ICSH) and of FSH necessary for normal sexual development in casesof hypogonadism or delayed puberty, and is also useful in the treatmentof cryptorchidism. Furthermore, the FSH released by the administrationof the decapeptide stimulates the development of germinal cells in thetestes, and the decapeptide is useful in the treatment of nonpsychogenicimpotence. And psychogenic impotence as can because of its possibleeffect on C.N.S.

When the decapeptide, preferably in the form of an acid addition salt,is employed in human medicine, it is administered systemically, eitherby intravenous, subcutaneous, or intramuscular injection, or bysublingual, nasal, or vaginal administration, in compositions inconjunction with a pharmaceutically acceptable vehicle or carrier.

For administration by the nasal route as drops or spray it is preferredto use the decapeptide in solution in a sterile aqueous vehicle whichmay also contain other solutes such as buffers or preservatives, as wellas sufficient quantities of pharmaceutically acceptable salts or ofglucose to make the solution isotonic. Doses by the intranasal routerange from 0.1 to 50 mcg/kg, or preferably 0.5 to 10 mcg/kg.

The decapeptide may also be administered as nasal or vaginal powders orinsufflations. For such purposes the decapeptide is administered infinely divided solid form together with a pharmaceutically acceptablesolid carrier, for example a finely divided polyethylene glycol("Carbowax 1540"), finely divided lactose, or preferably for vaginaladministration, very finely divided silica ("Cab-O-Sil"). Suchcompositions may also contain other excipients in finely divided solidform such as preservatives, buffers, or surface active agents.

For sublingual or vaginal administration it is preferred to formulatethe decapeptide in solid dosage forms such as sublingual tablets orvaginal inserts or suppositories with sufficient quantities of solidexcipients such as starch, lactose, certain types of clay, buffers, andlubricating, disintergrating, or surface-active agents, or withsemi-solid excipients commonly used in the formulation of suppositories.Examples of such excipients are found in standard pharmaceutical texts,e.g. in Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa., 1970.

The dosage of the decapeptide will vary with the form of administrationand with the particular patient under treatment. Generally, treatment isinitiated with small dosages substantially less than the optimum dose ofthe compound. Thereafter, the dosage is increased by small incrementsuntil the optimum effect under the circumstances is reached. In general,the decapeptide obtained by the process is most desirably administeredat a concentration level that will generally afford effective release ofLH and of FSH without causing any harmful or deleterious side effects,and preferably at a level that is in a range of from about 0.1 mcg. toabout 100 mcg. per kilogram body weight, although as aforementionedvariations will occur. However, a dosage level that is in the range offrom about 0.5 mcg. to about 5 mcg per kilogram body weight is mostdesirably employed in order to achieve effective results.

It si often desirable to administer the decapeptide continuously overprolonged periods of time in long-acting, slow-release, or depot dosageforms. Such dosage forms may either contain a pharmaceuticallyacceptable salt of the compound having a low degree of solubility inbody fluids, for example one of those salts decribed below, or they maycontain the decapeptide in the form of a water-soluble salt togetherwith a protective carrier which prevents rapid release. In the lattercase, for example, the decapeptide may be formulated with anon-antigenic partially hydrolyzed gelatin in the form of a viscousliquid; or it may be adsorbed on a pharmaceutically acceptable solidcarrier, for example zinc hydroxide, and may be administered insuspension in a pharmaceutically acceptable liquid vehicle; or thedecapeptide may be formulated in gels or suspensions with a protectivenon-antigenic hydrocolloid, for example sodium carboxymethylcellulose,polyvinylpyrrolidone, sodium alginate, gelatine, polygalacturonic acids,for example, pectin, or certain mucopolysaccharides, together withaqueous or nonaqueous pharmaceutically acceptable liquid vehicles,preservatives, or surfactants. Examples of such formulations are foundin standard pharmaceutical texts, e.g. in Remington's PharmaceuticalSciences, cited above. Long-acting, slow-release preparations of thedecapeptide may also be obtained by microencapsulation in apharmaceutically acceptable coating material, for example gelatine,polyvinyl alcohol or ethyl cellulose. Further examples of coatingmaterials and of the processes used for microencapsulation are describedby J. A. Herbig in "Encyclopedia of Chemical Technology", Vol. 13, 2ndEd., Wiley, New York, 1967, pp. 436-456. Such formulations, as well assuspensions of salts of the decapeptide which are only sparingly solublein body fluids, are designed to release from about 0.1 mcg to about 50mcg of the hormone per kilogram body weight per day, and are preferablyadministered by intramuscular injection. Alternatively, some of thesolid dosage forms listed above, for example certain sparinglywater-soluble salts or dispersions in or adsorbates on solid carriers ofsalts of the decapeptide, for example dispersions in a neutral hydrogelor a polymer of ethylene glycol methacrylate or similar monomerscross-linked as described in U.S. Pat. No. 3,551,556 may also beformulated in the form of pellets releasing about the same amounts asshown above and may be implanted subcutaneously or intramuscularly.

Alternatively, slow-release effects over prolonged periods of time mayalso be obtained by administering the decapeptide obtained by theprocess of this invention as an acid addition salt in an intra-vaginaldevice or in a temporary implant, for example a container made of anon-irritating silicone polymer such as a polysiloxane, e.g. "Silastic",or of a neutral hydrogel of a polymer as described above, possessing therequired degree of permeability to release from about 0.1 mcg. to about50 mcg per kilogrm body weight per day. Such intra-vaginal or implantdosage forms for prolonged administration have the advantage that theymay be removed when it is desired to interrupt or to terminatetreatment.

Process

In selecting a particular side chain protective group to be used in thesynthesis of the present decapeptide, the following rules should befollowed: (a) the protective group must be stable to the reagent andunder the reaction conditions selected for removing the α-aminoprotective group at each step of the synthesis, (b) the protective groupmust retain its protecting properties (i.e., not be split off undercoupling conditions), and (c) the side chain protective group must beremovable upon the completion of the synthesis containing the desiredamino acid sequence under reaction conditions that will not alter thepeptide chain.

With reference to R⁷, suitable protective groups include (1) aliphaticurethan protective groups illustrated by t-butyloxycarbonyl,diisopropylmethoxycarbonyl, biphenylisopropyloxycarbonyl,isopropyloxycarbonyl, t-amyloxycarbonyl, ethoxycarbonyl,allyloxycarbonyl; (2) cycloalkyl urethan type protective groupsillustrated by cyclopentyloxycarbonyl, adamantyloxycarbonyl,d-isobornyloxycarbonyl, cyclohexyloxycarbonyl; nitrophenylsulfenyl,tritylsulfenyl, α,α-dimethyl-3,5-dimethyoxybenzyloxycarbonyl and trityl.The preferred α-amino protective group for R⁷ are selected from thegroup consisting of t-butyloxycarbonyl, cyclopentyloxycarbonyl,t-amyloxycarbonyl, d-isobornyloxycarbonyl, o-nitrophenylsulfenyl,biphenylisopropyloxycarbonyl, andα,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl.

The decapeptide of this invention is prepared using solid phasesynthesis. The synthesis is commenced from the C-terminal end of thepeptide using an α-amino protected resin. Such a starting material isprepared by attaching an α-amino protected glycine to a benzhydrylamineresin, a chloromethylated resin or a hydroxymethyl resin, the formerbeing preferred. The preparation of a benzhydrylamine resin is describedby P. Rivaille, et al., Helv. Chim. Acta, 54, 2772 (1971) and thepreparation of the hydroxymethyl resin is described by M. Bodanszky andJ. T. Sheehan, Chem. Ind (London) 38, 1597 (1966). A chloromethylatedresin is commercially available from Bio Rad Laboratories, Richmond,California. In using the benzhydrylamine resin an amide anchoring bondis formed with the α-amino protected glycine as follows: ##STR2##

This permits the C-terminal amide function to be obtained directly afterthe amino acid sequence in the synthesis is completed by cleaving offthe resin support of the linked peptide to form the glycine amide at theC-terminal protion of the desired decapeptide. In this instance the useof hydrogen fluoride for cleaving off the resin support also removes theside chain protective groups to give the decapeptide of this invention.

When the other resins are used, the anchoring bond is the benzylestergroup as illustrated hereinbefore. In this instance a convenientprocedure for converting the linked protected peptide to the C-terminalamide is to ammonolize the protected peptide off the resin and thenremove the protective groups of the resulting amide by treatment withsodium and liquid ammonia or by hydrogen fluoride cleavage. Analternative procedure would be to cleave by transesterification with alower alkanol, preferably methanol or ethanol, in the presence oftriethylamine and then convert the resulting ester into an amide andsubsequently deprotect as described above. See also J. M. Steward and J.D. Young, "Solid Phase Peptide Synthesis", W. H. Freeman & Co., SanFrancisco, 1969, pp. 40-49.

More specifically, in an embodiment of the present invention an α-aminoprotected glycine, preferably t-butyloxycarbonylglycine, is coupled tobenzhydrylamine resin with the aid of the carboxyl group activatingcompound, preferably, dicyclohexylcarbodiimide. Following the couplingof the α-amino protected glycine to the resin support, the α-aminoprotecting group is removed such as by using trifluoroacetic acid inmethylene chloride, trifluoroacetic acid alone or hydrochloric acid indioxane. The deprotection is carried out at a temperature between about0° C and room temperature. Other standard cleaving reagents andconditions for removal of specific α-amino protecting groups may be usedas described by E. Schroder and K. Lubke, "The Peptides", Vol. 1,Academic Press, New York, 1965, pp. 77-75. After removal of the α-aminoprotecting group, the remaining α-amino protected amino acids arecoupled step-wise in the desired order to obtain the decapeptide. Eachprotected amino acid is introduced into the solid phase reactor in abouta three-fold excess and the coupling is carried out in a medium ofmethylene chloride or mixtures of dimethylformamide in methylenechloride. In cases where incomplete coupling occurred the couplingprocedure is repeated before removal of the α-amino protecting group,prior to the coupling of the next amino acid to the solid phase reactor.The success of the coupling reaction at each stage of the synthesis ismonitored by the ninhydrin reaction as described by E. Kaiser, et al.,Analyt. Biochem. 34, 595 (1970).

After the desired amino acid sequence has been synthesized, the peptideis removed from the resin support by treatment with a reagent such ashydrogen fluoride which not only cleaves the peptide from the resin butalso cleaves all remaining side chain protecting groups and the α-aminoprotecting group (if present) on the pyroglutamic acid residue to obtaindirectly the decapeptide in the case where the benzhydrylamine resin wasused.

Where a chloromethylated resin is used the peptide may be separated fromthe resin by transesterification with a lower alkanol, preferablymethanol or ethanol, after which the recovered product ischromatographed on silica gel and the collected fraction subjected totreatment with ammonia to convert the lower alkyl ester, preferably themethyl or ethyl ester, to the C-terminal amide. The side chainprotecting groups are then cleaved by procedures described above, forexample by treatment with sodium in liquid ammonia or by hydrogenfluoride.

Although a solid phase synthesis of the decapeptide of this invention isdisclosed herein, the preparation of the decapeptide also can berealized by classical methods. For example, by following the procedureof H. U. Immer et al., U.S. Pat. No. 3,835,108, issued September 10,1974 but substituting Ser-Tyr-D-Phe-NHNHBoc for Ser-Tyr-Gly-NHNHBoc, thedecapeptide also is obtained (via(pyro)-Glu-His-Trp-Ser-Tyr-D-Phe-NHNH₂).

The following Example illustrate further this invention.

EXAMPLE 1

L-Pyroglutamyl-L-histidyl(tosyl)-L-tryptophyl-L-seryl(benzyl)-L-tyrosyl(2-bromo-benzyloxycarbonyl)-D-phenylalanyl-L-leucyl-L-arginyl(tosyl)-L-prolylglycylbenzhydrylamineresin (R⁶ -(pyro)-Glu-His-(N^(lm)-R₅)-Trp-Ser(R⁴)-Tyr(R³)-D-Phe-Leu-Arg(N^(G) -R²)-Pro-Gly-A; R² = Tos,R³ = 2-Br-Cbz, R⁴ = Bzl, R⁵ = Tps, R⁶ = H and A = benzhydrylamineresin).

Benzhydrylamine resin (1.25 g, 1.0 mmole) is placed in the reactionvessel of a Beckman Model 990 automatic peptide synthesizer programmedto carry out the following wash cycle: (a) methylene chloride; (b) 33%trifluoroacetic acetic in methylene chloride (2times for 2.5 and 25minutes each); (c) methylene chloride; (d) ethanol; (e) chloroform; (f)10% triethylamine in chloroform (2 times for 25 minutes each); (g)chloroform; (h) methylene chloride.

The washed resin is then stirred with t-butyloxycarbonyl glycine (525mg, 3.0 mmoles) in methylene chloride and dicyclohexylcarbodiimide (3.0mmoles) is added. The mixture is stirred at room temperature (22°-25° C)for 2 hours and the amino acid resin is then washed successively withmethylene chloride (3 times), ethanol (3 times), and methylene chloride(3 times). The attached amino acid is deprotected with 33%trifluoroacetic acid in methylene chloride (2 times for 2.5 and 25minutes each and then steps (c) through (h) as described in the abovewash cycle are preformed.

The following amino acids (3.0 mmoles) are then coupled successively bythe same cycle of events: t-Boc-L-protine; t-Boc-L-arginine(Tos);t-Boc-L-leucine; t-Boc-D-phenylalanine; t-Boc-L-tyrosine(2 Br-Obz);t-Boc-L-serine(Bzl); t-Boc-L-tryptophan; t-Boc-L-histidine(Tos);L-(pyro)-glutamic acid.

The completed decapeptide resin is washed with methylene chloride (3times) followed by methanol (3 times) and dried under reduced pressurewhereupon 100% of the theoretical weight gain is obtained.

The benzhydrylamine resin used in this example is a commerciallyavailable resin (1% cross linked, Bachem Inc., Marina del Rey,California).

EXAMPLE 2

L-Pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-phenylalanyl-L-leucyl-L-arginyl-L-prolylglycinamide

Removal of protecting groups and cleavage of the decapeptide from theresin is carried out by treatment of 1.5 g of material with hydrogenfluoride (24 ml) and anisole (6 ml) at 0° C for 30 minutes. The hydrogenfluoride is removed under reduced pressure and the anisole removed bywashing with ethyl acetate.

The crude peptide is purified by gel filtration on a column (2.5 × 100cm) of Sephadex G-25 (a fine grade, chemically modified cross-linkeddextran) by elution with 2 molar acetic acid and fractions shown tocontain a major peak by UV absorption at 280 nm were pooled andevaporated to dryness.

The residual oil was applied to a column (2.5 × 100 cm) of Sephadex G-25(fine), previously equilibrated with the lower phase followed by theupper phase of n-butanol; acetic acid; water (4:1:5) solvent system.Elution with the upper phase gives a major peak and fractions from thispeak were pooled and concentrated to dryness. The residue waslyophilized from 0.2 N acetic acid to give [D-Phe⁶ ]-LH-RH as a fluffywhite powder (158 mg); [α]_(D) ²⁵ - 57.5° (C = 0.55, O in HoAc).

The product was homogeneous by thin layer chromatography in fourseparate solvent systems when loads of 20-30 mcg were applied and spotsvisualized by exposure to iodine vapour followed by Ehrlich reagent. Thefollowing Rf values were obtained.

1-butanol; acetic acid: water (4:1:5: upper phase), 0.14; ethyl acetate:pyridine: acetic acid: water (5:5:1:3), 0.68; 2-propanol: l M aceticacid (2:1), 0.41; 1-butanol: acetic acid: water: ethyl acetate(1:1:1:1), 0.47.

Amino acid analysis gave: glu, 1.01; His, 0.97; Trp, 0.90; Ser, 0.92;Tyr, 1.00; Phe, 0.96; Leu, 1.00; Arg, 1.02; Pro, 0.95; Gly, 1.00; NH₃,1.00.

We claim:
 1. A compound of the formulaR⁶ -(pyro)-Glu-His(N^(lm)-R⁵)-Trp-Ser(R⁴)-Tyr(R³)-D-Phe-Leu-Arg-(N^(G) -R²)-Pro-Gly-R¹ in whichR¹ is amino or O-(lower alkyl), R² is a protective group for theN.sup.δ, N.sup.ω, and N.sup.ω^(') nitrogen atoms of arginine selectedfrom the group consisting of tosyl, nitro benzyloxycarbonyl andadamantyloxycarbonyl; R³ is a protective group for the hydroxyl oftyrosine selected from the group consisting of 2-bromobenzyloxycarbonyl,benzyl, acetyl, tosyl, benzoyl, t-butyl, tetrahydropyran-2-yl, trityl,2,4-dichlorobenzyl and benzyloxycarbonyl; R⁴ is a protective group forthe hydroxyl group of serine and is selected from the group definedhereinbefore for R³ ; R⁵ is a protective group for the imidazolenitrogen atoms of histidine selected from the group of dinitrophenyl,tosyl, 2,2,2-trifluoro-1-benzoyloxycarbonylaminoethyl and2,2,2-trifluoro-t-butyloxycarbonylaminoethyl; and R⁶ is hydrogen or anα-amino protective group selected from the group consisting oft-butyloxycarbonyl, benzyloxycarbonyl, cyclopentyloxycarbonyl,t-amyloxycarbonyl and d-isobornyloxycarbonyl.
 2. The compound of claim 1in which R¹ is amino.
 3. The compound of claim 1 wherein R¹ is amino, R²is tosyl, R³ is 2-bromo-benzyloxycarbonyl, R⁴ is benzyl, R⁵ is tosyl andR⁶ is hydrogen.
 4. The compoundL-pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-phenylalanyl-L-leucyl-L-arginyl-L-prolylglycinamide,or a non-toxic, pharmaceutically acceptable addition salt thereof.
 5. Acompound selected from the group consisting of R⁶-(pyro)-Glu-His-(N^(1m) -R⁵)-Trp-Ser(R⁴)-Tyr(R³)-D-Phe-Leu-Arg(N^(G)-R²)-Pro-Gly-A, R⁷ -His(N^(1m)-R⁵)-Trp-Ser(R⁴)-Tyr-(R³)-D-Phe-Leu-Arg(N^(G) -R²)-Pro-Gly-A, R⁷-Trp-Ser(R⁴)-Tyr-(R³)-D-Phe-Leu-Arg(N^(G) -R²)-Pro-Gly-A, R⁷-Ser(R⁴)-Tyr(R³)-D-Phe-Leu-Arg(N^(G) -R²)-Pro-Gly-A, R⁷-Tyr(R³)-D-Phe-Leu-Arg-(N^(G) -R²)-Pro-Gly-A, and R⁷-D-Phe-Leu-Arg(N^(G) -R²)-Pro-Gly-A, in whichR² is a protective groupfor the N.sup.δ, N.sup.ω, and N.sup.ω^(') nitrogen atoms of arginineselected from the group consisting of tosyl, nitro, benzyloxycarbonyland adamantyloxycarbonyl; R³ is a protective group for the hydroxyl oftyrosine selected from the group consisting of2-bromo-benzyloxycarbonyl, benzyl, acetyl, tosyl, benzoyl, t-butyl,tetrahydropyran-2-yl, trityl, 2,4-dichlorobenzyl and benzyloxycarbonyl;R⁴ is a protective group for the hydroxyl group of serine and isselected from the group defined hereinbefore for R³ ; R⁵ is a protectivegroup for the imidazole nitrogen atoms of histidine selected from thegroup of dinitrophenyl, tosyl,2,2,2-trifluoro-1-benzoyloxycarbonylaminoethyl and2,2,2-trifluoro-t-butyloxycarbonylaminoethyl; and R⁶ is hydrogen or anα-amino protective group and R⁷ is an α-amino protective group, and A isselected from the class consisting of: ##STR3##
 6. The compoundaccording to claim 5 in which A is a benzhydrylamine resin.
 7. Thecompound of claim 5 having the formula R⁶ -(pryo)-Glu-His-(N^(1m)-R⁵)-Trp-Ser(R⁴)-Tyr(R³)-D-Phe-Leu-Arg(N^(G) -R²)-Pro-Gly-A in which R²is tosyl, R³ is 2-bromo-benzyloxycarbonyl, R⁴ is benzyl, R⁵ is tosyl, R⁶is hydrogen and A is a benzhydrylamine resin.